PROJECT SUMMARY The cell surface of pathogenic bacteria contains many key virulence factors that are used to interface with the host. Cell surface polymers also contain the molecular signatures recognized by the innate immune system to activate a defensive response, and surface molecules or their biogenesis pathways serve as important targets for many of our most effective vaccine and antibiotic therapies. A better understanding of the mechanisms responsible for bacterial surface assembly will therefore impact virtually all areas of pathogenesis research and inform the development of new treatments for infections. Although some aspects of cell surface assembly can be inferred from studies of non-pathogenic organisms, results from the models will never be entirely predictive. Departures from the model are likely to be especially pronounced for pathogens like Streptococcus pneumoniae (Sp) that adopt a different (ovoid) morphology and grow via distinct mechanisms from rod-shaped organisms like Escherichia coli and Bacillus subtilis where studies of cell surface assembly have traditionally been investigated. Sp is a major cause of life-threatening disease in young children and older adults, and the incidence of drug-resistant infections with this organism is on the rise. The efficacy of the polyvalent Sp vaccine is also declining due to the emergence of strains with altered surface polysaccharides that escape vaccine-induced immunity. It is therefore important to identify new ways of disabling Sp growth. To do so, we have initiated a program to investigate cell surface assembly in Sp that leverages the joint expertise of the Rudner and Bernhardt laboratories in cell wall biogenesis, gram-positive biology, microscopy, biochemistry, and genetics. Importantly, our approach is not limited to the characterization of homologs of well-studied cell morphogenesis factors from the rod-shaped model organisms. Instead we are taking advantage of forward genetic screens powered by modern sequencing methods to discover new players and biological mechanisms involved in Sp growth. Our preliminary genetic analyses have uncovered two novel regulators of the penicillin- binding proteins (PBPs) of Sp. These are the first set of factors identified in gram-positive bacteria that control the activity of these critical cell wall synthases. The first aim of the project will investigate the mechanism by which these factors modulate PBP activity and connect the function of these enzymes with other components of the morphogenetic system. In addition to controlling PBP activity, proper surface assembly also requires the regulation of enzymes that cleave the cell wall. The factors governing the activity of these enzymes are poorly understood in all bacteria. Our second aim will build on promising results where we have identified a regulatory role for surface polymers called lipoteichoic acids (LTAs) in controlling the activity of the cell wall hydrolase LytA responsible for Sp cell lysis following beta-lactam treatment. Overall, our results promise to uncover new ways of either blocking cell wall assembly or triggering autolysis for therapeutic development.